1. Field of the Invention
The present invention relates generally to methods which monitor binding of a compound to a target of interest inside a mammalian cell by increasing the stability of the target of interest and therefore its abundance, or number of molecules, in a cell. Further, the invention also relates to screening small molecules as inhibitors of target-compound binding in a cell.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described.
Binding between and among various molecules such as proteins is central to biological processes essential for normal cell functioning. Cellular pathways, such as signal transduction, apoptosis, cell and growth proliferation, gene regulation, require a series of interactions and bindings between and among specific proteins. Following transcription and translation, proteins assume their functional shape or confirmation through synchronized protein folding. The correct protein folding is essential for proteins to carry out their desired functions and expression. Proteins which do not undergo folding or do not fold in the requisite manner will have a short half-life in a cell and will be discarded by the cell as the degradation system of the cell will recognize it as an improper protein. Properly folded proteins will have a longer half-life and participate in binding events or other required cellular functions. Thus, stability of proteins is essential for protein expression and functioning in a cell, which is achieved through correct structure and participating in binding events leading to complex formation.
Protein binding occurs through special characteristic sequences or surfaces which are known as interacting interfaces. E.g. in case of protein-protein interactions these interfaces are known as ‘hotspots’ and constitute tight fitting regions which are generally characterized by complementary pockets scattered through the central region of the interface and enriched in structurally conserved residues (Hubbard and Argos Cavities and packing at protein interfaces. Protein Sci 1994; 3: 2194-2206). These pockets are classified as “complementary” (Binkowski et al, Inferring functional relationships of proteins from local sequence and spatial surface patterns J. Mol Biol 2003; 332: 505-526) because there is a large complementarity both in shape and in the juxtraposition of hydrophobic and hydrophilic hot spots, with buried charged residues (Arkin and Wells Small molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat Rev Drug Discov 2004; 3: 301-317). The “hotspots” are the essential amino acid sequences to participate in binding and to carry out physiological functions.
Other aspect of protein function is the allosteric modulation and has long been recognized as a general and widespread mechanism for the control of protein function. Modulators bind to regulatory sites distinct from the active site (hotspots) on the protein, resulting in conformational changes that may profoundly influence protein function and is a classical control mechanism of protein behavior. As described above, proteins are in native state as they undergo allosteric modulation.
Of particular importance (and exemplified below) are protein kinases, which participate in the process of phosphorylation, whereby they transfer phosphate groups from high-energy donor molecules, such as ATP to specific substrates. Protein phosphorylation is one of the most significant signal transduction mechanisms by which intercellular signals regulate crucial intracellular processes such as ion transport, cellular proliferation, and hormone responses. Kinases are the key regulators of cell function that constitute one of the largest and most functionally diverse gene families. They direct the activity, localization and overall function of many proteins and almost all cellular processes. Protein kinases can be regulated by activator proteins, inhibitor proteins, ligand binding to regulatory subunits, cofactors and phosphorylation by other proteins or by themselves (autophosphorylation). Kinases are prominent in signal transduction and co-ordination of complex functions such as the cell cycle. Thus, it makes kinases a potential target class for therapeutic interventions in many disease states such as cancer, diabetes, inflammation, and arthritis and represent as much as thirty percent of all protein targets under investigation by pharmaceutical companies.
A number of methods have been developed and used to study cellular binding events. Physical methods such as protein affinity chromatography, affinity blotting, immunoprecipitation and cross-linking however, either detect interactions that are so weak or else detect nearest neighbors which may not be in direct contact. Library-based methods such as protein probing and phage display also have certain intrinsic limitations. Two-hybrid system is only limited to proteins that can be localized to the nucleus, which may prevent its use with certain extracellular proteins. Translocation assays are very time consuming and expensive, as they require a confocal fluorescence system and are plagued by artifacts such as intrinsic fluorescence from combinatorial libraries and background fluorescence. However, none of the method is easy to perform, can be done in high throughput mode are amenable to studying disruption of constitutive biological complexes and identifying interaction interface.
Further, systems for protein stabilization and destabilization assays have also been developed. Studies by Stankunas et al (Stankunas K., Bayle H., Havranek J J., Wandless T J., Baker D., Crabtree G R., Gestwicki J E. Rescue of degradation-prone mutants of the FK506-Rapamycin binding (FRB) protein with chemical ligands; ChemBioChem, 2007; 8(10): 1162-1169) have shown the rescue of degradation prone mutants of FRB protein with chemical ligands. However, owing to the structure and half-life of luciferase in a cell it may interfere with the functioning of the target protein and also limits the availability of ligands to stabilize the fusion protein for the binding assays.
Earlier studies and assays have focused on large peptides and natural products as the primary compound classes capable of modulating cellular bindings. However a growing number of evidence has suggested that small molecules also modulate the interactions responsible for biological complexes (Toogood, Inhibition of protein-protein association by small molecules: approaches and progress. J Med Chem 2002, 45:1543-1558. Berg T, Modulation of protein-protein interactions with small organic molecules. Angew Chem Int Ed Engl 2003, 42:2462-2481). There are a number of advantages in using small molecules as inhibitors owing to higher metabolic stability, bioavailability, easier synthesis and great solubility among others.
The present invention is exemplified by use of enzyme fragment complementation. First shown in prokaryotes, enzyme fragment complementation (EFC) using beta-galactosidase (β-gal) is based on the complementation of enzyme fragments to form an active enzyme See, e.g., Ullman et al., J. Mol. Biol. 24:339-43 (1967); Ullman et al., J. Mol. Biol. 32:1-13 (1968); Ullman et al., J. Mol. Biol. 12:918-23 (1965). These systems have important advantages including low level expression of the test proteins, generation of signal as a direct result of the interaction, and enzymatic amplification. As a result, they are highly sensitive and physiologically relevant assays. See, e.g., Blakely et al., Nat. Biotechnol. 18:218-22 (2000).